DNA replication must occur with extraordinary accuracy to permit proper organismal development and to maintain cellular homeostasis. Inevitably, DNA damage occurs due to intrinsic and exogenous factors such as irradiation and chemical exposure. DNA damage is particularly dangerous during the DNA replication process because it can cause DNA replication forks to "stall", leading to even more severe DNA damage or chromosomal instabilities. Cells have evolved complex mechanisms to repair DNA damage, as well as cell cycle checkpoints to inhibit DNA replication and mitosis while providing an opportunity for DNA repair. We have recently found that the intra-S checkpoint regulates DNA synthesis at replication forks, inhibiting replication upon checkpoint activation and permitting replication fork restart upon deactivation of the checkpoint. This direct connection between DNA synthesis at the fork and checkpoint regulation strongly suggests that the intra-S checkpoint coordinates replication fork restart and DNA repair mechanisms, collectively referred to as "DNA-Damage Tolerance" (DDT) pathways, with DNA replication. We propose to study the involvement of DDT in the restart of replication forks that have stalled in response DNA damage, as well as the regulation of DDT by the intra-S checkpoint. The Specific Aims of this proposal are to: 1) Characterize replication fork dynamics in response to DNA damage, 2) Investigate the role of the intra-S checkpoint pathway in replication fork restart, 3) Perform molecular and genetic analysis of DDT mechanisms in replication fork restart. Lay Abstract: Cancers, developmental defects, and other genetic disorders are frequently caused by mutation of DNA. Mutations, though rare, sometimes occur during DNA replication, the process that produces an exact copy of an organism's copies entire DNA sequence to produce new cells. Because the uncontrolled growth of cancer cells depends on DNA replication, many chemotherapies act by disrupting DNA replication, which unfortunately have side-effects for normal cell growth. We have developed new technologies to study DNA replication at replication forks where the DNA is actually copied. This study will investigate the cellular processes that regulate replication forks to prevent mutations or minimize their potential damage. These studies have the potential of providing improved methods for the detection and treatment of cancer. PUBLIC HEALTH RELEVANCE: Project Narrative This proposal will investigate the regulation and molecular mechanisms of replication fork restart after DNA damage in S. cerevisiae. Our studies will focus on the role of intra-S checkpoint factor Rad53 in controlling the activity of replication forks encountering DNA damage and coordination of DNA-damage tolerance mechanisms that function in replication restart and DNA repair. These checkpoint and DNA repair mechanisms are critical to the prevention of genome instabilities that can lead to the development of cancers and other genetic disorders.